Difference between revisions of "Team:Bielefeld-CeBiTec/Project/PRIA"
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<h3>Immobilized Repressor</h3> | <h3>Immobilized Repressor</h3> | ||
<p>Our approach was built on Fusionproteins of the repressor proteins with cellulose binding domains (CBD). By adding these proteins to paper, we hoped to immobilize them. </p> | <p>Our approach was built on Fusionproteins of the repressor proteins with cellulose binding domains (CBD). By adding these proteins to paper, we hoped to immobilize them. </p> | ||
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| + | <figcaption>Illustration of PRIA with repressor protein bound to a cellulose matrix. The signal measured is generated by the release of plasmid DNA.</figcaption> | ||
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Revision as of 16:15, 9 September 2015
Plasmid Repressor Interaction Assay
A cell free detection system based on two purified components
Introduction
Our second approach to cell free detection of analytes is an approach based on an immobilized repressor that binds to a plasmid. When the analyte binds the repressor, it changes its conformation and releases the DNA. This release is to be measured. We called this approach Plasmid Repressor Interaction Assay. The advantage over the cell extract is, that this approach does not required transcription or translation of a reporter protein and therefore the signal can be measured much faster. Furthermore, it works with just two purified components, thereby further minimizing the risk of releasing GMOs into the wild.
Aim
Our aim was to develop a sensor based on the disruption of the repressor-plasmid bond. Since paper based systems are increasing in populartiy, because they are cheap and easily renewable, we wanted to establish a paper-based test strip.
Strategies
We started our experiments with a good characterized model system for repression and derepression: the lac operon and its repressor LacI. LacI immobilized on a Ni NTA column delivered the first proof of concept. There are two possible options for the transfer of this system from an impractial Ni NTA column to paper. The first one would be to immobilize the repressor on Paper, the second is to immobilize the DNA on Paper
Immobilized Repressor
Our approach was built on Fusionproteins of the repressor proteins with cellulose binding domains (CBD). By adding these proteins to paper, we hoped to immobilize them.
Immobilized DNA
Since the immobilization on ssDNA onto paper has been reported before (Araújo et al.) we aimed at using this system for the immobilization of dsDNA and combine it with an approach based on the measurement of the disruption of the binding of an GFPtagged repressor to immobilzied DNA (Siddiki et al.)
Outview
We established the proof of concept for PRIA, and we showed that the immobilization of dsDNA on paper is feasible. We have sfGFPtagged repressor proteins, so the next step would be to bring these aspects together and optimize a protocol that allows the establishment of the protein DNA complex on paper.
References
- Araújo et al., 2012, Activated Paper Surfaces for the rapid hybridization of DNA through Capillary Transport
- Siddiki et al., 2011, Solid Phase Biosensors for Arsenic or Cadmium Composed of A trans Factor and cis Element Complex